This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The repertoire of peptides bound by major histocompatability complexes (MHC) determines the fate of important biological outcomes such as the cellular defense and immune responses to foreign invaders or in some aberrant cases reactivity to self antigens. Detailed analyses of naturally processed peptides from class I and II MHC molecules have revealed certain general features, for example the lengths of naturally processed peptides selected by class I MHC molecules are usually restricted to 8-10-mers whereas those selected by class II MHC molecules are longer and more variable, usually between 14-24-mer. More importantly, detailed analyses of naturally processed peptides have addressed key issues regarding the specificity of peptide-selection among closely related MHC molecules, which was not evident in prior studies involving synthetic peptide libraries. In this analyses, we sought to identify the nature and motif of peptides that are selected by the class I MHC molecule, H-2Kd. We selected H-2Kd because it may play a significant role in several areas of disease including: responses against viral and bacterial pathogens, autoimmune diabetes, tumor immunology, and the efficacy of peptide-based tumor vaccines. Results from our analyses of large numbers of peptides selected by H-2Kd have firmly established the peptide-binding motif. Although a large fraction of naturally selected peptides were 8 to 10 amino acids in length, there were a significant fraction that were of longer length, some as long as 18-mers. Binding studies demonstrated that while the short peptides bound with higher affinity and formed long-lived peptide-MHC complexes, the longer peptides bound weakly and had a fast dissociation rate. Trimming the long peptides did not improve binding interactions and conversely extending the 9-mer naturally processed epitopes did not decrease binding. Finally, the mode of binding of the longer peptides was investigated. Results from these experiments demonstrate that the Naturally processed peptides selected by H-2Kd varied in length from 8-mers to 18-mers ? although most peptides were 8-10 amino acids, there was a significant fraction that were 10 amino acids in length. The immunological significance of these peptides is currently under investigation.